Albright's hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by short stature, brachydactyly, subcutaneous ossifications, and reduced expression or function of the alpha subunit of the stimulatory G protein of adenylyl cyclase (G(s)alpha). In most AHO patients deficient G(s)alpha activity is associated with generalized target organ resistance to hormones that act via stimulation of adenylyl cyclase. This form of the disorder is termed pseudohypoparathyroidism type Ia (PHP la). By contrast, other patients with G(s)alpha deficiency have only subclinical evidence of hormone resistance, and are considered to have the related disorder pseudopseudohypoparathyroidism(pseudoPHP). Thus AHO has emerged as an important paradigm of the functional consequences of impaired signal transduction through the adenylyl cyclase system. We have a well characterized group of AHO patients with G(s)alpha deficiency, including subjects with PH? type la and subjects with pseudoPHP. This research project will investigate the molecular basis for G(s)alpha deficiency in patients with AHO. Our preliminary studies indicate that the primary defect in AHO is within the structural gene for G(s)alpha. Our approach over the next 5 years involves molecular characterization of defects in the G(s)alpha gene which lead to defective function or expression of the G(s)alpha protein. We will analyze genomic DNA from subjects with AHO by restriction endonuclease analysis to identify deletions, insertions, or rearrangements within the G(s)alpha gene. To identify small (e.g. point) mutations we will use denaturing gradient gel electrophoresis to analyze specific exons and exon-intron junctions of the G(s)alpha gene that have been amplified by polymerase chain reaction. Expression of the abnormal G(s)alpha gene will be characterized by analysis of G(s)alpha protein and mRNA in cells from the patient. To determine the functional consequences of changes in the primary structure of the mutant G(s)alpha protein, mutant G(s)alpha proteins will be expressed in S49 cyc cells, which genetically lack endogenous G(s)alpha, and the signalling functions of normal and mutated G(s)alpha will be compared. The molecular delineation of mutations in the structural gene for G(s)alpha in patients with AHO will reveal defects that result in impaired expression or function of G(s)alpha protein. These "accidents of nature" will ultimately provide insights into the function of specific structural domains of the G(s)alpha protein. We predict that characterization of additional defects within the G(s)alpha gene in patients with AHO will further broaden our understanding of the molecular events of neurohormonal signal transduction from cell surface receptors to adenylyl cyclase.